The renin-angiotensin system is a major regulator of blood pressure and has been implicated in the pathogenesis of hypertension. Angiotensinogen (AGT), the initiating substrate of the system has been genetically linked to hypertension. Two AGT gene variants in the promoter region of the gene have been associated with hypertension and hypertension sequelae. Both variants A-20C and G-217A exhibit increased transcriptional activity in cultured angiotensinogen-expressing cells. We provide compelling preliminary data suggesting that these two variants are functional and physiologically significant. We also provide compelling preliminary data implicating the differential binding of specific transcription factors to the -20 and -217 polymorphisms as the mechanistic explanation for differences in transcriptional activity. In this competitive renewal, we will test the hypothesis that the level of human AGT expression in liver and extra-hepatic tissues, in particular adipose tissue, is dictated by differential transcription factor binding to polymorphic sites in the AGT promoter. We further hypothesize that differential expression of AGT in tissues plays an important role in the regulation of arterial pressure, water and electrolyte homeostasis. We proposed four specific aims to test these novel hypotheses. Aim 1 is to directly test the hypothesis that mice carrying the -20C allele of AGT when compared to mice carrying the -20A allele exhibit increased expression of AGT, increased recruitment of USF1/2 to the AGT promoter in chromatin, higher levels of circulating AGT and Ang-II, all resulting in elevated arterial pressure. Aim 2 will test the hypothesis that AGT expression is regulated by the differential binding of USF1/2 to A-20C, and C/EBP2 and GR to G-217A, recruitment of these factors to the AGT promoter in chromatin, association with transcriptional co-activators, and by modifications to histone proteins which control the conformation and transcriptional state of chromatin. Aim 3 will test the hypotheses that the level of mouse AGT expression is decreased in USF1-deficient and USF-2 deficient mice, the levels of -20C AGT are reduced to a greater degree than -20A AGT in USF1- and USF2-deficient mice, and the normal induction of AGT expression in response to a high fat diet in adipose tissue is blunted in USF1-deficient mice. Finally, in aim 4 we will add a new and exciting translational component to test the hypothesis that the level of AGT expression in human adipose tissue correlates with enhanced transcription factor binding to the A-20C and G-217A alleles in chromatin. These studies will allow us to address fundamental mechanisms by which polymorphisms linked to hypertension cause differential expression of AGT in cells and tissues;will provide novel and unique data on the mechanism of AGT regulation in an important and accessible human tissue (i.e. adipose tissue), and will examine the importance of allele-specific AGT production on arterial pressure. The aims are based on our productivity, generation of robust preliminary data supporting both methods and hypotheses, and the realization of powerful genetic reagents to manipulate the human and endogenous mouse RAS in vivo. PUBLIC HEALTH RELEVANCE: The Angiotensinogen gene is the initiating substrate of the renin-angiotensin system and a major regulator of blood pressure. Angiotensinogen has been implicated in the pathogenesis of hypertension, and has been genetically linked to human essential hypertension. In this proposal, we will define the molecular mechanisms by which two variants in the angiotensinogen gene cause hypertension through studies of human cell lines, transgenic mouse models and human tissues.